This proposal will determine how metformin and salicylate activate AMP kinase (AMPK), a key step in 'turning off' hepatic glucose production. AMPK exists as a heterotrimeric complex comprised of a catalytic ? subunit and regulatory ? and ? subunits. Binding of AMP to the ? subunit causes activation of the kinase by promoting phosphorylation at T172 on the ??subunit. AMPK activation in response to pharmacological agents has considerable potential to reverse the metabolic abnormalities associated with type 2 diabetes (T2DM). Previous studies have focused on metformin and aspirin activation of AMPK for the treatment of patients with T2DM; however, both of these agents are generally tested at concentrations that are either much higher than maximally achievable therapeutic concentrations or are known to cause toxicity in humans. Therefore, this study aims to test these drugs on primary mouse hepatocytes and a mouse model of impaired fasting glucose at low, therapeutic concentrations. First, the effect of low doses of metformin and aspirin on the AMPK heterotrimeric complex will be explored by expressing a FLAG-tagged AMPK?1 subunit in primary hepatocytes and immunoprecipitating this protein (using an anti-FLAG antibody) from cell lysates after metformin or aspirin treatment. To test whether these drugs affect formation of the AMPK heterotrimeric complex directly or indirectly, FLAG-tagged AMPK subunits will be generated. Primary hepatocytes will be used to express subunits and then treated with metformin or aspirin in both a dose-dependent and time-dependent protocol. This will be followed by immunoprecipitation using an anti-?1 antibody. We hypothesize that metformin and aspirin at low concentrations will increase phosphorylation of AMPK?1 via different mechanisms, as seen in preliminary studies. These drugs will then be tested at therapeutic levels in vivo using a mouse model which disinhibits cAMP-PKA signaling in the mouse liver by ablating the hepatic PKA regulatory subunit 1A (prkar1a) yielding L-?prkar1a mice. This model has been shown to mimic the effect of increased glucagon associated with the increased insulin resistance of T2DM. Control and diet induced obesity (DIO) prkar1a and L-?prkar1a mice will be treated with metformin and aspirin alone and in combination. We anticipate the two agents, when used together, will show synergy in decreasing hepatic glucose production; salicylate by blocking cAMP phosphorylation (reversing the cAMP effect, analogous to the glucagon effect seen in T2DM) allowing metformin to promote assembly of the complex that had been blocked by cAMP stimulation. Given that diabetic patients manifest fasting hyperglycemia associated with insulin resistance, the combination of both low dose salicylate and metformin may enhance AMPK assembly, activation and subsequent treatment efficacy without producing unwanted toxicities associated with high doses of these drugs. This knowledge would be especially helpful in the design of future therapeutic interventions for the insulin resistance associated with T2DM.